The goal of the CIP/CCR Frederick Flow Cytometry Core is to provide the highest quality instrumentation, education, and analytic support to the CCR investigators and assist in furthering their research goals. During the last year, the following improvements were made to the core to better support CCR investigators and their staff. Training Program Enhancement In support of the National Cancer Institute's mission to provide training and support for cancer researchers, the Flow Core Staff have adapted their training program to clarify the training presentations, improve and ensure user safety, and provide consistently available support. The training program includes the sub-units on principles of multiparametric flow cytometry, how to perform analyses on samples, the care, set up, and use of the flow cytometers, and to analyze their data on multiple software platforms. During 2019, the CIP/CCR Frederick Flow Core staff trained 31 new scientists (45% increase over the number trained in FY 2018). Furthermore, Flow Core Staff now offers training in post-acquisition analyses programs (such as FlowJo). This addition, strongly implemented this year, provides investigators and their staff with additional support in analyzing their own data effectively. This includes preparation of data for publication, and overall reduces the wait-time for investigators so they do not have to depend fully on Flow Core Staff availability to analyze their data on their behalf. Also, during 2019, the Flow Core Staff also designed (for several laboratories) multiparameter tumor infiltrating leukocyte identification panels and trained multiple users in the principles of multiparameter panel design. During the last year, the CIP/CCR Frederick Flow Core evaluated available core cytometer usage and needs. Based on user feedback, two (new) analyzers were added to better meet the needs of its researchers. Item 1: Miltenyi MACSQuant 16. This cytometer is capable of high-throughput analyses and can to acquire samples automatically from either 96 well plate or racked tube formats. The CIP/CCR Frederick Flow Core users have consistently requested an analysis platform which would permit acquisition of samples directly from 96 well plates (or tubes in racks). To meet this need, the flow core requested and received a Miltenyi MACSQuant-16 analyzer. This cytometer is capable of reading samples labeled with (up to) 14 different fluorochromes and can automatically load samples from 96 well plates. The 14-parameter system will be able to meet the needs of many of the Core users. It will also reduce the time required by investigators in sample manipulation as well as reduce the quantity of plastic consumables required for each experiment, thus decreasing both labor and supply costs. Item 2: BD Symphony A5 Cytometer. This instrument is an analyzer capable of high-level multiparameter analyses. This year, we also acquired a Becton Dickinson Symphony A5, which can measure fluorochromes in 28 different parameters (plus 2 light scatter parameters). The currently available analyzers in the core can evaluate a maximum of 16 fluorochrome parameters + 2 light scatter parameters. The need for the additional parameters is based on two key factors which continue to evolve in cell analysis: 1. A steady increase in the number of markers used to accurately identify cell types, subtypes, and activation status, and 2. the need to evaluate samples with small cell numbers (for example, Tumor Infiltrating Leukocytes (TIL), or circulating Cancer Stem Cells (CSC)). Historically, due to a limited number of parameters available for measurement on existing machines, samples had to be subdivided into smaller portions to allow measurement of all the needed parameters. With the acquisition of the new cytometer, the number of available parameters increases from 16 to 28, which will allow scientists to perform analyses on samples with small or limiting cell numbers. Effectively, this will make some analyses possible which were previously impossible due to small sample size.